傣药帕崩板激活肠肝轴AMPK治疗酒精性肝病的机制研究

Dai medicine Gynura procumbens is a folk remedy used for disease associated with alcoholic liver disease (ALD).We found that phenylpropionic acids from Gynura procumbens (PG) activated AMPK in the liver and intestinal mucosa (gut-liver axis) and had a protective effect on the early phase of ALD in mice. PG reduced the serum endotoxin level and inhibited the liver steatosis. The gut-liver axis theory for ALD is consistent with the traditional ‘Yagai’ theory in Dai medicine, means dredging the middle pan and relieving alcoholism. In the present study, ethanol liquid diets were used to induce chronic alcoholic fatty liver and fibrosis in rats. The protective effect of PG on those models will be evaluated by gut and liver function, as well as histological examination. H4IIEC3 cells will be used to mimic alcoholic fatty liver in vitro. The protective effect of PG on lipid metabolism, including fatty acid synthesis, β-oxidation and VLDL secretion will be investigated by radiolabeling techniques. AMPK and its downstream gene in lipid metabolism will be studied to reveal the mechanism. Caco-2 cell monolayer will be used to mimic alcohol-induced intestinal barrier leakiness in vitro. The stability of Caco-2 barrier will be evaluated by transepithelial electrical resistance measurement and FITC-D4 permeation determination. The AMPK-(Afadin, ATP)-ZO-1 pathway will be studied to reveal the mechanism of how PG protects the tight junction and inhibits the translocation of gut-derived endotoxin to the liver. The present study will supply new case to Dai medicine as well as new therapeutic target for drug development in ALD, and develop more useful drugs for ALD treatment in clinic.

傣药帕崩板在民间用于治疗酒精性肝病相关疾病。我们前期筛选发现，帕崩板苯丙酸类化合物（PG）在肝脏和肠黏膜（肠肝轴）激活AMPK，抑制肝脏脂肪蓄积，降低血中内毒素水平，对抗早期酒精性肝病，符合傣医雅解理论：通中盘，解酒毒。本项目拟以酒精液体饲料建立大鼠慢性酒精性脂肪肝和肝纤维化模型，通过肝、肠功能和组织学检查，探讨PG的药理作用；以H4IIEC3细胞建立肝细胞酒精损伤模型，通过同位素标记检测脂质代谢（脂肪酸合成、β-氧化和脂质分泌）情况，围绕AMPK及其下游脂质代谢各关键因子，探讨PG抑制肝脏脂肪蓄积的机制；以Caco-2细胞建立肠黏膜上皮细胞酒精损伤模型，通过跨膜电阻和荧光标记检测细胞间通透性，围绕AMPK-(Afadin, ATP)-ZO-1通路，探讨PG保护肠黏膜，阻止肠腔内毒素移位入肝的机制。预期成果将为傣医药的发展提供新的实证支持，为酒精性肝病新药研发提供新的靶点，丰富临床用药。

傣药帕崩板在民间用于治疗酒精性肝病相关疾病。我们前期筛选发现，帕崩板苯丙酸类化合物（PG）在肝脏和肠黏膜（肠肝轴）激活AMPK，抑制肝脏脂肪蓄积，降低血中内毒素水平，对抗早期酒精性肝病（ALD），符合傣医雅解理论：通中盘，解酒毒。本项目的主要研究内容有三部分：①将醇提物分散在水中，依次用石油醚、乙酸乙酯、正丁醇萃取，并用急性模型证实正丁醇部位是醇提物的有效部位；将正丁醇部位上聚酰胺柱，水-60%乙醇-95%乙醇洗脱，并用急性模型证实60%乙醇洗脱部位是正丁醇部位的有效部位，HPLC分析其含有13.6%的绿原酸；用慢性模型证实了帕崩板醇提物、60%乙醇洗脱部位、绿原酸均对酒精引起的脂肪肝有治疗作用。并发现该作用是通过MAPK-SREBP1依赖和非依赖途径实现的；利用含鱼油的Lieber-Decarli酒精液体饲料加上每周一次大剂量酒精灌胃制造了新的酒精性脂肪性肝炎模型，并证实大剂量鱼油通过抑制Kupffer细胞M2型极化而加重肝炎的毒理机制；②然后我们利用制备液相将帕崩板60%乙醇洗脱部位分成了绿原酸类部位和非绿原酸部位，在酒精性脂肪性肝炎模型上进行了药效研究，发现绿原酸和非绿原酸部位协同发挥作用；③以Caco-2细胞建立肠黏膜上皮细胞酒精损伤模型，发现绿原酸稳定ZO-1、Occludin-1表达，保护肠黏膜。帕崩板多组分协同作用于肝脏和肠黏膜AMPK，治疗ALD，这与傣医雅解理论吻合，是良好的ALD 防治候选药物。